Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae

doi:10.1073/pnas.0635171100

Genome-wide analysis of mRNA translation profiles in Saccharomycescerevisiae

^*Department of Biochemistry, Stanford University, Stanford, CA 94305-5307; ^†The Howard Hughes Medical Institute, Stanford, CA 94305-5428; and ^‡Department of Statistics, Stanford University, Stanford, CA 94305-4065

Edited by Nicholas R. Cozzarelli, University of California, Berkeley, CA, and approved January 23, 2003 (received for review August 26, 2002)

Abstract

We have analyzed the translational status of each mRNA in rapidly growing Saccharomyces cerevisiae. mRNAs were separated by velocity sedimentation on a sucrose gradient, and 14 fractions across the gradient were analyzed by quantitative microarray analysis, providing a profile of ribosome association with mRNAs for thousands of genes. For most genes, the majority of mRNA molecules were associated with ribosomes and presumably engaged in translation. This systematic approach enabled us to recognize genes with unusual behavior. For 43 genes, most mRNA molecules were not associated with ribosomes, suggesting that they may be translationally controlled. For 53 genes, including GCN4, CPA1, and ICY2, three genes for which translational control is known to play a key role in regulation, most mRNA molecules were associated with a single ribosome. The number of ribosomes associated with mRNAs increased with increasing length of the putative protein-coding sequence, consistent with longer transit times for ribosomes translating longer coding sequences. The density at which ribosomes were distributed on each mRNA (i.e., the number of ribosomes per unit ORF length) was well below the maximum packing density for nearly all mRNAs, consistent with initiation as the rate-limiting step in translation. Global analysis revealed an unexpected correlation: Ribosome density decreases with increasing ORF length. Models to account for this surprising observation are discussed.

Footnotes

↵ § To whom correspondence may be addressed. E-mail: herschla{at}cmgm.stanford.edu or pbrown{at}cmgm.stanford.edu.
This paper was submitted directly (Track II) to the PNAS office.
↵ ¶ It is not clear, however, whether there are secondary effects on translation after cycloheximide addition. To minimize the possible effects we immediately cooled and harvested the cells after cycloheximide addition. To investigate the possibility of additional initiation events after cycloheximide addition, we quantified [³⁵S]Met incorporation prior to and subsequent to cycloheximide addition. Initiation was inhibited at least 99%, but determining a limit to the maximal number of initiation events after cycloheximide addition was not possible, because the intracellular pool size of unlabeled Met and background are not known. Further varying the cycloheximide concentrations (0.01–0.25 mg/ml) had no effect on the overall polysome profile, providing no indication of complicating secondary effects or additional initiation events under our preparation conditions. The conclusions described herein would be unaffected even in the extreme case that each mRNA was to have a ribosome added subsequent to cycloheximide treatment. Finally, effects of cycloheximide on translation termination have been reported in mammalian systems, which give considerable differences in the polysomes response to varying concentrations of cycloheximide (refs. 30–32 and data not shown). Mapping studies suggest that the termination effect is not significant for the results presented herein (Y.A., F. E. Boas, P.O.B., and D.H., unpublished data).
↵ ‖ mRNAs potentially sedimenting with the 40S subunit (fractions 3 and 4) were not considered to be translationally active, because they cannot be distinguished from mRNAs associated with other large RNA–protein complexes. Thus, the ribosome occupancy values may underestimate the number of molecules engaged in some step of translation. On the other hand, some of the mRNAs associated with a single ribosome could have accumulated after the cycloheximide treatment; rapid coding was employed to minimize such effects. We estimate, based on microarray polysome profiles and measurements of mRNA abundance (9), that 14% of all mRNA molecules are associated with a single ribosome.
↵ ** This selection biases the sample toward mRNAs with prominent peaks rather than broad distributions across the gradient (e.g., compare ACT1 and PDC1 in Fig. 2 B). The distribution width may reflect underlying differences in translational usage. For example, mRNAs with varying translational rates through the cell cycle would appear more broadly distributed in polysome profiles of mRNA obtained from unsynchronized cells.
↵ ‡‡ This holds for most models. However, one can envision extreme cases in which densities will vary between two mRNAs with similar initiation, elongation, and termination rates. For example, if termination were severely rate-limiting, then ribosomes would tend to accumulate at the 3′ end of the message, and mRNAs with shorter coding sequences would tend to have higher ribosome densities than mRNAs with longer coding sequences even if the rates of initiation, elongation, and termination were the same for the two.